Different mechanisms could explain the origin and heterogeneity of CSC such as (i) differentiation arrest (stem cells), (ii) dedifferentiation (mature cells) and (iii) transdifferentiation (bone marrow stem cells). It is conceivable that all 3 mechanisms may be corrupted by oncogenic events, resulting in an assortment of CSC and explaining their heterogeneity. Defining and characterizing this heterogeneity is of vital importance for understanding CSC biology, and for effective therapeutic translation. Our most recent results in this project include: (1)Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of HCC cells. Our strategy employed a 3 day treatment of established and primary human HCC derived cell lines grown as monolayer at various cell densities with the DNMT1 inhibitor Zebularine (ZEB) followed by a 3D culture to identify cells endowed with selfrenewal potential. Differences in self renewal, gene expression, tumorigenicity and metastatic potential of spheres at generations G1 to G5 were examined. Transient ZEB exposure produced differential cell densitydependent responses. In cells grown at low density, ZEB caused a remarkable increase in selfrenewal and tumorigenicity associated with longlasting gene expression changes characterized by a stable overexpression of cancer stem cell related and key epithelial mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast, at high cell density, ZEB caused a gradual decrease in selfrenewal and tumorigenicty, and upregulation of apoptosis and differentiation related genes. A permanent reduction of DNMT1 protein using shRNA mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, WRL68 and HepG2 hepatoblastoma cells expressing low DNMT1 basal levels also possessed a high self renewal irrespective of cell density or ZEB exposure. Spheres formed by low density cells treated with ZEB or shDNMT1A displayed a high molecular similarity which was sustained through consecutive generations, confirming the essential role of DNMT1 depletion in the enhancement of cancer stem cell properties.Conclusion: These results identify DNA methylation as a key epigenetic regulatory mechanism determining the pool of cancer stem cells in liver cancer and possibly other solid tumors.(2)Human primary liver cancer is classified into biologically distinct subgroups based on cellular origin. Liver cancer stem cells (CSCs) have been recently described. We investigated the ability of distinct lineages of hepatic cells to become liver CSCs and the phenotypic and genetic heterogeneity of primary liver cancer. We transduced mouse primary hepatic progenitor cells, lineage committed hepatoblasts, and differentiated adult hepatocytes with transgenes encoding oncogenic HRas and SV40LT. The CSC properties of transduced cells and their ability to form tumors were tested by standard in vitro and in vivo assays and transcriptome profiling. Irrespective of origin, all transduced cells acquired markers of CSC/progenitor cells, side populations, and selfrenewal capacity in vitro. They also formed a broad spectrum of liver tumors, ranging from cholangiocarcinoma to hepatocellular carcinoma, which resembled human liver tumors, based on genomic and histologic analyses. The tumor cells coexpressed hepatocyte (hepatocyte nuclear factor 4alpha, progenitor/biliary (keratin 19, epithelial cell adhesion molecule, A6), and mesenchymal (vimentin) markers and showed dysregulation of genes that control the epithelial mesenchymal transition. Gene expression analyses could distinguish tumors of different cellular origin, indicating the contribution of lineage stage dependent genetic changes to malignant transformation. Activation of cMyc and its target genes wasrequired to reprogram adult hepatocytes into CSCs and for tumors to develop. Stable knockdown of cMyc in transformed adult hepatocytes reduced their CSC properties in vitro and suppressed growth of tumors in immunodeficient mice. From these data we conclude that any cell type in the mouse hepatic lineage can undergo oncogenic reprogramming into a CSC by activating different cell type specific pathways. Identification of common and cell of origin specific phenotypic and genetic changes could provide new therapeutic targets for liver cancer.(3)The standard therapy for advanced hepatocellular carcinoma (HCC) is sorafenib, with most patients experiencing disease progression within 6 months. Label retaining cancer cells (LRCC) represent a novel subpopulation of cancer stem cells (CSC). Our objective was to test whether LRCC are resistant to sorafenib. We tested human HCC derived LRCC and non LRCC before and after treatment with sorafenib. LRCC derived from human HCC are relatively resistant to sorafenib. The proportion of LRCC in HCC cell lines is increased after sorafenib while the general population of cancer cells undergoes growth suppression. We show that LRCC demonstrate improved viability and toxicity profiles, and reduced apoptosis, over non LRCC. We show that after treatment with sorafenib, LRCC upregulate the CSC marker aldehyde dehydrogenase 1 family, wingless type MMTVintegration site family, cell survival and proliferation genes, and downregulate apoptosis, cell cycle arrest, cell adhesion and stem cells differentiation genes. This phenomenon was accompanied by non uniform activation of specific isoforms of the sorafenib target proteins extracellular signal regulated kinases and v aktmurine thymoma viral oncogene homologue (AKT) in LRCC but not in non LRCC. A molecular pathway map for sorafenib treated LRCC is proposed. Our results suggest that HCC derived LRCC are relatively resistant to sorafenib. Since LRCC can generate tumours with as few as 10 cells, our data suggest a potential role for these cells in disease recurrence. Further investigation of this phenomenon might provide novel insights into cancer biology, cancer recurrence and drug resistance with important implications for the development of novel cancer therapies based on targeting LRCC.